Potentiation of Imipramine-Induced Anti-hyperalgesic and Anti-Nociceptive Effects by Citicoline in the Sciatic Nerve Ligated Mice

Background: Peripheral neuropathic pain is a result of damage/illness of the peripheral nerves. The mechanisms caused by its pathophysiology are not completely understood. Methods: Imipramine is a tricyclic antidepressant that is sometimes used to treat neuropathic pain. Moreover, citicoline is considered a novel adjuvant for painful disorders such as neuropathic pain. So, a possible interaction between imipramine and citicoline on pain behavior was examined in nerve-ligated mice using tail-flick and hot plate tests. Results: The results indicated that induction of neuropathic pain by sciatic nerve ligation caused hyperalgesia in nerve-ligated mice. On the other hand, intraperitoneal (i.p.) administration of citicoline (50, 75, and 100 mg/kg), and imipramine (2.5 and 5 mg/kg) induced anti-hyperalgesic and anti-nociceptive effects in nerve-ligated mice. Furthermore, citicoline potentiated the anti-hyperalgesic and anti-nociceptive effects of imipramine when they were co-administrated in nerve-ligated mice. Interestingly, there was an additive effect between imipramine and citicoline upon induction of anti-hyperalgesic and anti-nociceptive effects in nerve-ligated mice. Conclusion: Therefore, it can be concluded that citicoline (as an adjuvant substance) enhanced the efficacy of imipramine for the modulation of pain behavior in nerve-ligated mice


Introduction
2][3][4] The understanding of a particular pathophysiological variation that contributes to the induction and preservation of neuropathic pain may provide a basis for the development of novel analgesic treatments for this illness.][10][11][12] Ligation of the sciatic nerve in mice is a relevant model for assessing nociceptive and emotional consequences of continued neuropathic pain. 13,14Although neuropathic pain often does not react to conservative analgesic therapies, non-conservative painkillers such as antidepressants can be effective. 15ntidepressants are often considered co-analgesics in chronic pain.7][18][19] The mechanism of action of imipramine in the improvement of neuropathic pain remains unclear, though it is prescribed as a robust reuptake inhibitor of serotonin and, to a lesser amount, norepinephrine. 18,20The mechanism is probable to vary from that in depression because analgesia with tricyclic antidepressants is frequently achieved at lower doses than required for antidepressant effects. 18Imipramine might induce an anti-nociceptive effect via activation of the serotonergic system. 21Imipramine may be administered either alone or in combination with other drugs for the treatment of neuropathic pains.For example, the cholinergic system is involved in the anti-nociceptive effect of imipramine. 22,23][26] Citicoline potentiates acetylcholine production. 26It is usually present in all cells.Exogenous citicoline simply crosses the blood-brain barrier and enhances the amount of choline available for acetylcholine synthesis as well as helps in rebuilding membrane phospholipid stores after depletion. 27iticoline prevents the neuronal phospholipid membrane breakdown and repairs the neuronal membrane after neuronal damage. 28Long-term treatment with citicoline increases endogenous mechanisms of neurogenesis and neuro-repair contributing to physical therapy and recovery. 291][32] This study examined a probable interaction between imipramine and citicoline on the control of neuropathic pain in nerve-ligated mice.
In the present study, we selected imipramine, a tricyclic antidepressant, and citicoline, a novel adjuvant, that might play a role in the modulation of neuropathic pain.A previous study has reported a cross-talk between imipramine and citicoline regarding the induction of antinociceptive effects. 33So, this research aimed to evaluate a possible interaction between imipramine and citicoline on the modulation of neuropathic pain in nerve-ligated mice.

Animals
Experiments were performed on male NMRI mice (6-8 weeks old, 20-25 g; Tehran University of Medical Sciences, Tehran, Iran) group-housed 3-4 per cage.The animals were maintained in a room with a controlled temperature of 23 ± 1 °C and a 12h/12h light/dark cycle (lights on 07:00 AM).Furthermore, food and water were available.Eight male mice were used for each experimental group.All experiments were done during the light period.The experimental techniques used in this research have been confirmed by the Ethics Committee of Tehran University of Medical Sciences (NIH publications No. 80-23).

Surgical Procedures
All surgeries were performed in sterile conditions and ketamine/xylazine anesthesia drugs (ketamine 50 mg/kg and xylazine 4 mg/kg, intraperitoneal (i.p.) injections).The common fiber of the right sciatic nerve was opened.Then, a 2-mm split section of the polyethylene tube was located near it.This induced an ipsilateral thermal hyperalgesia and a sustained ipsilateral mechanical allodynia. 13The shaved skin level was closed by a suture.The sham group underwent a similar surgery but without dissection of the sciatic nerve.The control group was only anesthetized.

Drug Treatment
We used 0.9% saline for the control injection and to dissolve all drugs.Imipramine (Ciba-Geigy, Switzerland) and citicoline sodium (Minoo, Tehran, Iran) were used in this investigation.All drug injections were done intraperitoneally (i.p.; 10 mL/kg).

Anti-nociception Measurement
Tail-Flick Test A tail-flick apparatus was used to evaluate the nociceptive response to thermal stimulation (Borj Sanat Company, Iran).Each animal was slightly wrapped in a soft towel and the dorsal surface of the tail from its distal end was located in the apparatus every 15 minutes (for 60 minutes ) after the drug/saline administration.The heat source and a timer were started rapidly via a pedal.Both were ended automatically by a tail movement, which exposed a photocell under the tail or by the experimenter at the end of a 10-second cut-off time.The cut-off time was set to avoid skin damage.The response time between the start of the heat stimulus and the removal of the tail from the heat source was recorded via a sensor as the tail-flick latency.Individual tail withdrawal latency was changed to the percentage of maximum possible effect (%MPE) by this formula: %MPE = [(test latency-baseline latency)/ (cut off latency-baseline latency)] × 100.For all data, the area under the curve (AUC) of %MPE vs. time was recorded from 0 to 60 min by the trapezoidal rule to analyze the overall magnitude and time of the effect for the tail-flick test.

Hot-Plate Test
Pain sensitivity in sciatic nerve ligated mice was also evaluated via a hot-plate test as clarified in prior research. 34or this test, mice were placed on a 52 ± 0.2 °C heated plate (Borj Sanat Company, Iran).The time to lick the forepaw, hind paw, or jump was measured.We prescribed a cut-off time of 60 s to avoid any tissue damage.

Experimental Design
This research consisted of four experiments.In experiment 1, the effect of sciatic nerve ligation on tail-flick latency and hot-plate latency was examined.In experiment 2, the influence of saline (10 mL/kg), and diverse dosages of citicoline (25, 50, 75, and 100 mg/kg, i.p.) on tail-flick and hot-plate latencies were assessed.In experiment 3, the effects of injection of saline alone (10 mL/kg) or diverse dosages of imipramine (1.25, 2.5, and 5 mg/kg, i.p.), as well as co-administration of diverse dosages of imipramine (1.25, 2.5, and 5 mg/ kg, i.p.) along with a low dose of citicoline (25 mg/kg; i.p.) were evaluated in the tail-flick and hot-plate tests.In experiment 4, the effects of co-injection of imipramine 2.5 mg/kg + citicoline 50 mg/ kg, and imipramine 1.25 mg/kg + citicoline 25 mg/kg, as well as imipramine 0.625 mg/kg + citicoline 12.5 mg/kg on pain-associated behaviors were examined.We used diverse groups of animals for the assessment of tail-flick and hotplate latencies and each animal was only used for one test.Ten minutes after drug injection, tail-flick, and hot-plate tests were done.Table 1 explains the experimental groups.

Statistical Analysis
Normal distribution of data was examined using the Kolmogorov-Smirnov test.The homogeneity of variances was assessed using Levene's test.The data were presented as mean ± standard error (SD) and 95% confidence interval (CI).The statistical analysis was done by one-way ANOVA and two-way ANOVA as well as Tukey's post hoc test.The data of tail-flick test were examined via two-way ANOVA and repeated measures.In the repeated measures ANOVA, normality and homogeneity of variance of residuals as well as sphericity were assessed.A P value less than 0.05 was considered for significant difference.
Furthermore, an isobolographic test was performed to define the interaction after the injection of two drugs.For this, the ED50 of each agent (2.5 mg/kg for imipramine and 50 mg/kg for citicoline) was examined via linear regression test and co-treatment of two drugs was administrated in fixed dosage ratio upon the ED50 amount.For drug co-treatment, theoretic ED50 is imipramine ED50/2 + citicoline ED50/2.Additionally, the experimental amount of drug combinations from the constant quantity determined was evaluated via the regression test (IBM SPSS Statistics, version 27 [IBM; Armonk, New York, USA]), after the experimental ED50 amount of the drug combinations was recorded (50% tailflick and hot-plate latencies).The statistical significance between the theoretical ED50 and experimental ED50 of the drug co-treatment was recorded via a one sample t-test.If experimental ED50 was significantly lower than theoretical ED50, a synergistic interaction between imipramine and citicoline could be detected.Nevertheless, there was no difference between them showing additive interaction. 33Differences with P values less than 0.05 among experimental groups at each level were determined as statistically different.

Effect of Sciatic Nerve Ligation on Tail-flick and Hot-Plate Latencies in Male Mice
The results were considered as mean difference estimates with 95% CI.Table 2 displayed the results of mean ± standard error and 95% CI.
Figure 1 indicates the effect of sciatic nerve ligation on tail-flick latency and hot-plate latency in male mice.Two-way ANOVA displayed no significant difference between sciatic nerve ligation and time intervals on %MPE [(time intervals effect F (1, 48) = 10.381,P < 0.001; sciatic nerve ligation effect F (2, 48) = 0.999, P = 0.400; time intervals × sciatic nerve ligation interaction F (2, 48) = 0.629, P = 0.599; Figure 1A].About the time interval effect and sciatic nerve ligation effect, Tukey's test showed that sciatic nerve ligation at the time intervals of 15, 30, 45 and 60 minutes increased %MPE in nerve-ligated mice in comparison with the control and sham groups.
As seen in Figure 1C, one-way ANOVA showed a significant effect of citicoline on hot-plate latency in nerve-ligated mice (mean difference: 9.812 and 95% CI: 8.729-10.895)[(F (5, 42) = 9.037, P < 0.001; Figure 2C] compared to the control and sham groups.Tukey's analysis demonstrated that injection of diverse dosages of citicoline (50, 75, and 100 mg/kg; i.p.) enhanced the pain threshold in comparison with the control and sham groups.These data suggested that citicoline induced anti-hyperalgesic and anti-nociceptive effects in nerve-ligated mice.

Discussion
Tail-flick and hot plate apparatuses are validated tests for the evaluation of pain.The difference between tail-flick and hot plate tests is that the tail-flick device assesses the nociceptive response mostly at the spinal level while the hot plate device assesses the supra-spinal response. 35nduction of neuropathic pain by ligation of the sciatic nerve is one of the best and most frequently used models of assessment of anti-hyperalgesic and anti-nociceptive properties of substances. 4,36Our research indicated that sciatic nerve ligation caused hyperalgesia in nerve-ligated mice in comparison with the control and sham groups.
Studies demonstrated that partial ligation of the sciatic nerve caused a highly reproducible syndrome in the mice, including a reduction in thermal and mechanical nociceptive thresholds and sustained alterations in neurotransmitter and receptor expression. 7,12Consistent with some studies on neuropathic pain, we also demonstrated hyperalgesia in nerve-ligated mice.
The current research showed that citicoline at doses of 50, 75, and 100 mg/kg reduced hyperalgesia.Here, citicoline dose-dependently induced anti-nociceptive effect following sciatic nerve ligation in mice.Citicoline has been demonstrated to prompt nerve regeneration after surgery in numerous in vitro investigations.Citicoline has been commonly used in clinical cases of central nervous system illnesses, for example, ischemic stroke, cognitive impairments, and glaucoma.Administration of citicoline has been revealed to play a role in improving motoric function and prompting the regeneration process of the impaired axons in a rat sciatic nerve damage model, showing its potential role in the treatment of peripheral nerve injury. 31,38,39According to our results, Kanat and colleagues indicated that citicoline exerted an anti-hyperalgesic effect in oxaliplatininduced neuropathic pain. 32Also, Emril et al reported that citicoline administration prevented peripheral neuropathic pain after sciatic nerve crush injury in rats. 31ollowing administration of citicoline, it is hydrolyzed to cytidine and choline, which leads to enhanced plasma and tissue concentrations of these metabolites. 40Citicoline stimulates the biosynthesis of structural phospholipids of the neuronal membranes, raises brain metabolism, and acts upon the levels of diverse neurotransmitters, for instance, norepinephrine and dopamine.Owing to its weird pharmacological properties and action mechanisms, citicoline has been described as a potential candidate agent for the treatment of several types of neurological disorders such as head trauma, cerebral vascular disease, and Alzheimer's disease. 41Recent preclinical investigations indicated that administration of citicoline elicited dose-and time-dependent anti-nociceptive and antihyperalgesic effects in behavioral models of neuropathic and inflammatory pain in rodents. 42,43These effects of citicoline might be mediated by an interaction between acetylcholine, norepinephrine, and dopamine receptors. 32n the next section of this research, we assessed the effect of the administration of imipramine alone on the modulation of neuropathic pain induced by sciatic nerve ligation in male mice.Our data exhibited that i.p. injection of imipramine dose-dependently enhanced %MPE and AUC of %MPE in the nerve-ligated mice, showing anti-hyperalgesic and anti-nociceptive effects.Tricyclic antidepressants such as imipramine have analgesic effects in diverse chronic pain disorders that are distinct from their antidepressant characteristics. 19,44][46] Different doses of imipramine induced a dose-dependent anti-nociceptive effect in the formalin test, 23,[46][47][48][49] tail-flick method, 22,33 and hot-plate test. 45The anti-nociceptive mechanism of antidepressants is still obviously detected.][23] Tricyclic antidepressants are used either alone or in combination with other substances to treat neuropathic pains, so we decided to examine a possible interaction between imipramine and citicoline as well as to explore the significance of imipramine doses (1.25, 2.5, and 5 mg/kg) on the modulation of neuropathic pain in nerveligated mice.Our findings exhibited that co-treatment of different doses of imipramine (1.25, 2.5, and 5 mg/kg) along with an ineffective dosage of citicoline (25 mg/kg) increased %MPE and AUC of %MPE in the nerve-ligated mice, suggesting that citicoline potentiated the antihyperalgesic and anti-nociceptive effects of imipramine.Interestingly, our data revealed an additive effect between imipramine and citicoline upon induction of antihyperalgesic and anti-nociceptive effects in nerve-ligated mice.As mentioned previously, the cholinergic system is involved in the anti-nociceptive effect of imipramine. 22,23e proposed that citicoline increased acetylcholine levels, 26 hence potentiated the antihyperalgesic and anti-nociceptive effects of imipramine.In this context, our previous research indicated that co-treatment of imipramine and citicoline induced an analgesic effect in intact male mice. 33

Conclusion
The anti-hyperalgesic and anti-nociceptive effects of imipramine and citicoline might be due to the enhancement of some neurotransmitters such as serotonin, acetylcholine, and norepinephrine.Furthermore, the encouraging results of the animal experiment may prompt further clinical assessment of the effects of imipramine and citicoline in the management of neuropathic pain.

1 A 2 A-nociceptive 3 A-nociceptive 4 A
Figure Panel Drug Treatments (i.p.) Effect on Tail-Flick Effect On Hot-Plate

Figure 1 .Figure 2 .
Figure 1.Influence of Sciatic Nerve Ligation on Pain Behavior in the Tail-Flick and Hot-Plate Latencies.Data are presented as mean ± SEM.Two-way ANOVA and repeated measures were performed for analysis of MPE% of the tail-flick test (A).One-way ANOVA and Tukey's post hoc test were performed for AUC of MPE% analysis (B) and hot-plate threshold (C).* P < 0.05, **P < 0.01, and *** P < 0.001 in comparison to the control group (n = 8)

Figure 3 .Figure 4 .
Figure 3. Influence of Administration of Imipramine Alone (1.25, 2.5, and 5 mg/kg) and Co-Administration of These Dosages Plus a Low Dose of Citicoline (25 mg/kg) on the Pain Behavior of Nerve-Ligated Mice Investigated in the Tail-Flick and Hot-Plate Tests (each drug was administered to nerve-ligated mice).Data are presented as mean ± S.E.M. Two-way ANOVA and repeated measures were performed for analysis of MPE% of the tail-flick test (A and B).One-way ANOVA followed by Tukey's post-hoc test was performed for AUC of MPE% analysis (C) and hot-plate threshold (D).* P < 0.05, ** P < 0.01 and *** P < 0.001 compared with control group.+ + P < 0.01 and + + + P < 0.001 compared to nerve-ligated group (n = 8)

Table 2 .
Results of Mean ± Standard Error and 95% CI